1. Field of the Invention
The present invention relates to a method for enhancing extracellular secretion of recombinant proteins, and more particularly relates to a method for enhancing extracellular secretion of recombinant proteins in Escherichia coli by co-expressing Thermobifida fusca (T. fusca) cutinase.
2. Description of the Related Art
Escherichia coli (E. coli) has a rapid growth rate and high protein expression level so that it has been considered as one of the most promising strain for industrial production of recombinant proteins. E. coli is able to express proteins in 3 ways: cytosolic production, periplasmic production and extracellular production. Compared with other two methods, extracellular production has significant advantages in both analytical and industrial applications because it does not require cell disruption and has almost no contamination from host proteins.
There are 5 secretory pathways for protein to translocate across the cell membrane in E. coli. However, due to their complicated components, regulatory mechanisms, and/or the low intrinsic secretion capabilities of these systems, E. coli is usually considered to be a poor secretor of proteins. It has been reported that the balance between expression level and secretion rate is a critical factor for extracellular production of recombinant proteins. To date, many strategies for improving the extracellular secretion of proteins in E. coli have been reported, especially the mechanism between transport chaperon protein and target proteins has been explored. Enhancing protein secretion process, constructing outer membrane leakage mutations as well as adding certain chemicals to enhance the permeability of cell membrane are commonly used methods.
Cutinase not only catalyzes the cleavage of the ester bonds of cutins, but also is capable of hydrolyzing soluble esters, insoluble triglycerides and a variety of polyesters. In addition to its hydrolytic activity, cutinase is also used in ester synthesis and transesterification. As a multifunctional enzyme, cutinase has potential in the food, chemical, textile, and other industries. Enzymes with cutinase activity have been found in both fungi and bacteria. To date, all the cutinase from microorganisms have been found to be secretory enzymes. Therefore, for the heterologous expression, signal peptides are usually used to mediate the secretion of recombinant cutinase. Using this approach, Fusarium solani cutinase has been expressed in a variety of host cells. The highest yield of extracellular protein 546 mg/L, was obtained in a 5 L bioreactor using an engineered Saccharomyces cerevisiae cellular system. Previously, we identified the open reading frame responsible for the expression of T. fusca cutinase, and secretory expression of the cutinase was performed by mediation of the pelB signal peptide in E. coli BL21 (DE3). More recently, we found that the mature form of T. fusca cutinase without a signal peptide was mainly secreted into the culture medium and only a small percentage of the enzyme is located in the periplasm and cytoplasm. We further found that the mature cutinase has phospholipase activity that leads to limited hydrolysis of phospholipids of cell membrane and thus increases membrane permeability. Based on this finding, the present invention provides a method for extracellular secretion of mature cutinase without mediation of a signal peptide and a method for enhancing extracellular secretion of recombinant proteins. Mature cutinase used herein refers to a full-length cutinase protein without a signal peptide.